Microwave ablation combined with PD-L1 blockade synergistically promotes Cxcl9-mediated antitumor immunity.

Although microwave ablation (MWA) is an important curative therapy in colorectal cancer liver metastasis, recurrence still occurs clinically. Our previous studies have shown that the expression of programmed cell death 1 ligand 1 (PD-L1) is upregulated following MWA, suggesting that MWA combined with anti-PD-L1 treatment can serve as a promising clinical therapeutic strategy against cancer. Using MWA-treated preclinical mice models, MWA combined with αPD-L1 treatment decreased tumor growth and prolonged overall survival (OS). Furthermore, through flow cytometry and single-cell RNA sequencing analysis, we determined that the MWA plus αPD-L1 therapy significantly suppressed CD8+ T cell exhaustion and enhanced their effector function. A significant increase in γ-interferon (IFN-γ) stimulated transcription factors, specifically Irf8, was observed. This enhancement facilitated the polarization of tumor-associated macrophages (TAM1s and TAM2s) through the nuclear factor-κB/JAK-STAT1 signaling pathway. Furthermore, the combination therapy stimulated the production of CXC motif chemokine ligand (CXCL9) by TAM1s and tumor cells, potentially increasing the chemotaxis of CD8 T cells and Th1 cells. Knocking out Cxcl9 in MC38 tumor cells or using CXCL9 blockade enhanced tumor growth of untreated tumors and shortened OS. Taken together, our study showed that blocking the IFN-γ-Cxcl9-CD8+ T axis promoted tumor progression and discovered a potential involvement of IRF8-regulated TAMs in preventing T cell exhaustion. Collectively, we identified that the combination of MWA with anti-PD-L1 treatment holds promise as a therapeutic strategy to rejuvenate the immune response against tumors. This merits further exploration in clinical studies.

Metabolism-dependent mutagenicity of two structurally similar tobacco-specific nitrosamines (N-nitrosonornicotine and N-nitrosoanabasine) in human cells, partially different CYPs being activating enzymes.

N-nitrosonornicotine (NNN) and N-nitrosoanabasine (NAB) are both tobacco-specific nitrosamines bearing two heterocyclic amino groups, NAB bearing an extra -CH2- group (conferring a hexa- rather than penta-membered cycle) but with significantly decreased carcinogenicity. However, their activating enzymes and related mutagenicity remain unclear. In this study, the chemical-CYP interaction was analyzed by molecular docking, thus the binding energies and conformations of NNN for human CYP2A6, 2A13, 2B6, 2E1 and 3A4 appeared appropriate as a substrate, so did NAB for human CYP1B1, 2A6, 2A13 and 2E1. The micronucleus test in human hepatoma (HepG2) cells with each compound (62.5-1000 µM) exposing for 48 h (two-cell cycle) was negative, however, pretreatment with bisphenol AF (0.1-100 nM, CYPs inducer) and ethanol (0.2% v:v, CYP2E1 inducer) potentiated micronucleus formation by both compounds, while CITCO (1 µM, CYP2B6 inducer) selectively potentiated that by NNN. In C3A cells (endogenous CYPs enhanced over HepG2) both compounds induced micronucleus, which was abolished by 1-aminobenzotriazole (60 µM, CYPs inhibitor) while unaffected by 8-methoxypsoralen (1 µM, CYP2A inhibitor). Consistently, NNN and NAB induced micronucleus in V79-derived recombinant cell lines expressing human CYP2B6/2E1 and CYP1B1/2E1, respectively, while negative in those expressing other CYPs. By immunofluorescent assay both compounds selectively induced centromere-free micronucleus in C3A cells. In PIG-A assays in HepG2 cells NNN and NAB were weakly positive and simply negative, respectively; however, in C3A cells both compounds significantly induced gene mutations, NNN being slight more potent. Conclusively, both NNN and NAB are mutagenic and clastogenic, depending on metabolic activation by partially different CYP enzymes.

Human CYP1A1-activated aneugenicity of aflatoxin B1 in mammalian cells and its combined effect with benzo(a)pyrene.

Aflatoxin B1 (AFB1) is the most toxic mycotoxin and a proven human carcinogen that requires metabolic activation, known by cytochrome P450 (CYP) 1A2 and 3A4. Previous evidence showed that AFB1 is activated by human recombinant CYP1A1 expressed in budding yeast. Yet, the toxicity, in particular the genotoxicity of the reactive metabolites formed from AFB1 remains unclear. Humans could be exposed to both AFB1 and benzo(a)pyrene (BaP) simultaneously, thus we were interested in their combined genotoxic effects subsequent to metabolic activation by CYP1A1. In this study, molecular docking of AFB1 to human CYP1A1 indicated that AFB1 is valid as a substrate. In the incubations with AFB1 in human CYP1A1-expressed microsomes, AFM1 as a marking metabolite of AFB1 was detected. Moreover, AFB1 induced micronucleus formation in a Chinese hamster V79-derived cell line and in a human lung epithelial BEAS-2B cell line, both expressing recombinant human CYP1A1, V79-hCYP1A1 and 2B-hCYP1A1 cells, respectively. Immunofluorescence of centromere protein B stained micronuclei was dominant in AFB1-treated BEAS-2B cells exposed to AFB1, suggesting an aneugenic effect. Moreover, AFB1 elevated the levels of ROS, 8-OHdG, AFB1-DNA adduct, and DNA breaks in 2B-hCYP1A1 cells, compared with those in the parental BEAS-2B cells. Meanwhile, AFB1 increased CYP1A1, RAD51, and γ-H2AX protein levels in 2B-hCYP1A1 cells, which were attenuated by the CYP1A1 inhibitor bergamottin. Co-exposure of AFB1 with BaP increased 8-OHdG, RAD51, and γ-H2AX levels (indicating DNA damage). In conclusion, AFB1 could be activated by human CYP1A1 for potent aneugenicity, which may be further enhanced by co-exposure to BaP.

Estimation of the cumulative risks from dietary exposure to cadmium, arsenic, nickel, lead and chromium in Guangzhou, China.

Heavy metals, such as cadmium and lead, are ubiquitously present as single substances and compounds in the environment. These substances have various and overlapping health effects. Consumption of contaminated foods is the main pathway of the human exposure, however, estimation of their dietary exposure in combination with health risk analysis, particularly at various endpoints, has rarely been reported. In this study, we integrated relative potency factor (RPF) analysis into the margin of exposure (MOE) model to evaluate the health risk of combined heavy metal (including cadmium, arsenic, lead, chromium, and nickel) exposure in the residents in Guangzhou, China, after quantifying the heavy metals in various food samples and estimating their dietary exposure. The results indicated that rice, rice products and leafy vegetables contributed primarily to the dietary exposure of all metals except arsenic, which exposed the population largely through consumption of sea-foods. With all the five metals contributing to nephro- and neurotoxicity, the 95% confidence limits of MOE for the residents were clearly below 1.0 in the 3∼6-year group, suggesting a recognizable risk to young children. This study provides substantial evidence for the non-negligible health risk in young children due to increased heavy metal exposure,at least on some toxicity targets.

Induction of clastogenesis and gene mutations by carbamazepine (at its therapeutically effective serum levels) in mammalian cells and the dependence on human CYP2B6 enzyme activity.

Carbamazepine (CBZ, an antiepileptic) is metabolized by multiple CYP enzymes to its epoxide and hydroxides; however, whether it is genotoxic remains unclear. In this study, molecular docking (CBZ to CYPs) and cytogenotoxic toxicity assays were employed to investigate the activation of CBZ for mutagenic effects, in various mammalian cell models. Docking results indicated that CBZ was valid as a substrate of human CYP2B6 and 2E1, while not for CYP1A1, 1A2, 1B1 or 3A4. In the Chinese hamster (V79) cell line and its derivatives genetically engineered for the expression of human CYP1A1, 1A2, 1B1, 2E1 or 3A4 CBZ (2.5 ~ 40 µM) did not induce micronucleus, while in human CYP2B6-expressing cells CBZ significantly induced micronucleus formation. In a human hepatoma C3A cell line, which endogenously expressed CYP2B6 twofold higher than in HepG2 cells, CBZ induced micronucleus potently, which was blocked by 1-aminobenzotriazole (inhibitor of CYPs) and ticlopidine (specific CYP2B6 inhibitor). In HepG2 cells CBZ did not induce micronucleus; however, pretreatment of the cells with CICTO (CYP2B6 inducer) led to micronucleus formation by CBZ, while rifampicin (CYP3A4 inducer) or PCB126 (CYP1A inducer) did not change the negative results. Immunofluorescent assay showed that CBZ selectively induced centromere-free micronucleus. Moreover, CBZ induced double-strand DNA breaks (γ-H2AX elevation, by Western blot) and PIG-A gene mutations (by flowcytometry) in C3A (threshold being 5 µM, lower than its therapeutic serum concentrations, 17 ~ 51 µM), with no effects in HepG2 cells. Clearly, CBZ may induce clastogenesis and gene mutations at its therapeutic concentrations, human CYP2B6 being a major activating enzyme.

Probabilistic risk assessment of dietary exposure to pentachlorophenol in Guangzhou, China.

Pentachlorophenol (PCP) is a ubiquitous environmental contaminant commonly existing as its sodium salt (NaPCP), which enters the human body primarily through long term but low-level dietary exposure. PCP contributes to chemical carcinogenesis and teratogenesis. In this study, the probabilistic risk of dietary exposure to PCP in Guangzhou citizens was investigated. In total, 923 food samples in the categories of pork, livestock (beef and lamb), poultry, offal, eggs, and freshwater fish (considered to be relatively susceptible to PCP contamination) were collected from various markets in Guangzhou and tested for PCP. Probabilistic risk assessment model calculations for PCP dietary exposure and margin of exposure (MOE) values were performed using @RISK software, based on a Monte Carlo simulation with 10,000 iterations. The overall detection rate of PCP (above 1 µg kg-1, the detection limit) was 19.9% (184/923), with an average of 7.9 µg kg-1. The highest rate of PCP detection, 28.2%, was in livestock (beef and lamb). The MOE value for dietary PCP exposure in general Guangzhou residents averaged 400, which was far below 5,000 (the borderline for judging a health risk). The lowest MOE value, 190, was observed in the 3- to-6-year old population and indicates a significant risk. In conclusion, this study suggests that PCP exposure in Guangzhou residents is of considerable health risk, especially for the pre-school young children.

Human CYP1B1 enzyme-mediated, AhR enhanced activation of aflatoxin B1 for its genotoxicity in human cells.

Aflatoxin B1 (AFB1) is a human procarcinogen known to be activated by cytochrome P450 (CYP) 1A2 and 3A4. In a previous study AFB1 caused chromosomal rearrangement in a yeast strain genetically engineered for stably expressing human CYP1B1. Yet, further verification of the effect of AFB1 in human cells, a potential role of the aryl hydrocarbon receptor (AhR), and CYP1B1-catalyzed AFB1 metabolism remain unidentified. In this study, a human hepatocyte (L-02) line and a human lymphoblastoid (TK6) cell line were genetically engineered for the expression of human CYP1B1, producing L-02-hCYP1B1 and TK6-hCYP1B1, respectively. They were exposed to AFB1 and analyzed for the formation of micronucleus and elevation of γ-H2AX (indicating double-strand DNA breaks); the metabolites formed by CYP1B1 from AFB1 after incubation of AFB1 with human CYP1B1 isoenzyme microsomes were determined by LC-MS. The results showed significantly more potent induction of micronucleus by AFB1 in L-02-hCYP1B1 and TK6-hCYP1B1 than in the parental (L-02 and TK6) cells, and the effects were reduced by (E)- 2,3',4,5'-tetramethoxystilbene, a specific CYP1B1 inhibitor. In the AFB1- CYP1B1 microsomes incubations AFM1, a known stable metabolite of AFB1, was detected. Moreover, in L-02 and TK6 cells, AFB1 apparently increased the protein levels of AhR, ANRT and CYP1B1, and caused the nuclear translocation of AhR and ARNT, the latter effect being blocked by BAY-218 (an inhibitor of AhR). In conclusion, this study indicates that human CYP1B1 is capable of metabolically activating AFB1 through the AhR signaling pathway.

Impact of Extended and Combined Exposure of Bisphenol Compounds on Their Chromosome-Damaging Effect─Increased Potency and Shifted Mode of Action.

Bisphenol (BP) compounds are important environmental pollutants and endocrine disruptors. BPs are capable of inducing DNA/chromosome breaks (clastogenesis, involved in carcinogenesis), which requires activation by human CYP1A1. We hypothesized that combined BPs and extended (from the standard two-cell cycle) exposure may enhance their genotoxicity via modulating CYP enzymes. In this study, individual and combined BPA/BPF/BPS/BPAF and a human hepatoma (HepG2) cell line were used for testing several genotoxicity end points. Exposing for a two-cell cycle period (48 h), each BP alone (0.625-10 µM) was negative in the micronucleus test, while micronucleus was formed under three- (72 h) and four-cell cycle (96 h) exposure; BP combinations further elevated the potency (with nanomolar thresholds). Immunofluorescence analysis of the centromere with formed micronucleus indicated that 48 h exposure produced centromere-negative micronucleus and phosphorylated histone H2AX (γ-H2AX) (evidencing clastogenesis), while extended (72 and 96 h) exposure formed centromere-positive micronucleus and phosphorylated histone H3 (p-H3) (indicating chromosome loss, i.e., aneugenesis); moreover, 1-aminotriabenzotriazole (CYP inhibitor) selectively blocked the formation of centromere-negative micronucleus and γ-H2AX, without affecting that of centromere-positive micronucleus and p-H3. This study suggests that the genotoxicity of BPs is potentiated by combined and extended exposure, the latter being specific for aneuploidy formation, a CYP activity-independent effect.

Triphenyl phosphate induces clastogenic effects potently in mammalian cells, human CYP1A2 and 2E1 being major activating enzymes.

As a new-type flame retardant and toxic substance, triphenyl phosphate (TPP) is a ubiquitous pollutant present even in human blood. TPP is transformed by human CYP enzymes to oxidized/dealkylated metabolites. The impact of TPP metabolism on its toxicity, however, remains unclear. In this study, the genotoxicity of TPP in several mammalian cell lines and its relevance to CYP/sulfortransferase (SULT) activities were investigated. The results indicated that TPP induced micronucleus formation at ≥1 µM concentrations in a human hepatoma (C3A, endogenous CYPs being substantial) cell line, which was abolished by 1-aminobenzotriazole (CYPs inhibitor). In cell line HepG2 (parental to C3A with lower CYP expression) TPP was inactive up to 10 µM, while pretreatment with ethanol (CYP2E1 inducer), PCB 126 (CYP1A inducer), or rifampicin (CYP3A inducer) led to micronucleus formation by TPP. In V79-Mz and V79-derived cells expressing human CYP1A1 TPP was inactive (up to 32 µM), and in cells expressing human CYP1B1, 2B6 and 3A4 it induced micronucleus weakly (positive only at 32 µM). However, TPP induced micronucleus potently in V79-derived cells expressing human CYP1A2, while this effect was drastically reduced by human SULT1A1 co-expression; likewise, TPP was inactive in cells expressing both human CYP2E1 and SULT1A1, but became positive with pentachlorophenol (inhibitor of SULT1) co-exposure. Moreover, in C3A cells TPP selectively induced centromere-free micronucleus (immunofluorescent assay), and TPP increased γ-H2AX (by Western blot, indicating double-strand DNA breaks). In conclusion, this study suggests that TPP is potently clastogenic, human CYP1A2 and 2E1 being major activating enzymes while SULT1A1 involved in detoxification.

Probabilistic Risk Assessment of Dietary Exposure to Cadmium in Residents of Guangzhou, China-Young Children Potentially at a Health Risk.

Cadmium (Cd) and its compounds are hazardous environmental pollutants with renal toxicity and human carcinogenicity, with ingestion of contaminated foods representing the major mode of exposure. There have been a number of reports evaluating the Cd content in various foods; however, regarding the actual risk posed by dietary cadmium exposure, only a few reports are available in which single point evaluation (less accurate than multiple point evaluation) was employed. In this study, we used a margin of exposure (MOE) model and @RISK software (for multiple evaluation) to evaluate Cd-related health risk in the local Guangzhou residents at varying ages, through a comparison between the estimated monthly exposures and the provisional tolerable monthly intake (0.025 mg/kg body weight (b.w.)), based on the Cd contents in various food categories available locally (a total of 3964 food samples were collected from each of the 13 districts of Guangzhou between 2015 and 2019), which were determined by using inductively coupled plasma mass spectrometry. In this study, Cd was detected in 69.6% of the samples (averaged 0.120 mg/kg), and rice and its products, leafy vegetables, bivalves, and shrimp and crabs contributed most to Cd exposure (8.63, 3.18, 2.79, and 1.48 ng/kg b.w./day, respectively). The MOE values demonstrated the following tendency: the younger age group, the lower MOE, and its 95% confidence range for the (youngest) 3~6 year old group started from 0.92, indicating a health risk of young children, while that for the other age groups were all above 1.0. Our preliminary findings warrant further clarification using biomarker assays in the relevant population.

Alleviation of doxorubicin-induced cardiomyocyte death through miR-147-y-mediated mitophagy.

Doxorubicin (DOX) is a commonly used antitumor drug. However, it may cause severe cardiotoxicity, apoptosis being a major change. A recent report indicates that miR-147 expression is decreased in the myocardium of a myocardial infarction model, suggesting a potential role of this miRNA in DOX-induced cardiomyocyte toxicity. In this study, freshly isolated neonatal pig cardiomyocytes were used; following transfection of a miR-147-y mimic, the cell death induced by DOX was alleviated, represented by augmented mitophagy [indicated by a decrease in P62, and increases in LC3, PINK1, parkin mRNA, LC3Ⅱ/Ⅰ, beclin-1, PINK1, and parkin including p-parkin (Ser65) protein expression], prohibited cell apoptosis as determined by TUNEL staining, and the suppression of caspase-3 transcription and cleaved caspase-3 translation. In cells transfected with an miR-147-y inhibitor, DOX-induced mitophagy was decreased, while apoptosis was increased. Additionally, RAPTOR gene silencing in cardiomyocytes exposed to DOX increased the rate of mitophagy and decreased that of apoptosis as compared with the treatment with DOX alone. Moreover, RAPTOR overexpression downregulated the rate of mitophagy and increased that of apoptosis in cells exposed to DOX. RAPTOR was confirmed as the target gene of miR-147-y based on the results of luciferase reporter gene assays and the opposite effects of the miR-147-y mimic and miR-147-y inhibitor on RAPTOR expression. In summary, our study suggests that miR-147-y mediates DOX-induced cardiomyocyte mitophagy while suppresses apoptosis by targeting RAPTOR, thus playing a protective role in DOX-induced cardiomyocyte damage.

Influence of aryl hydrocarbon receptor and sulfotransferase 1A1 on bisphenol AF-induced clastogenesis in human hepatoma cells.

Bisphenol compounds (BPs) are ubiquitously existing pollutants. Recent evidence shows that they may be activated by human CYP1A1 for clastogenic effects; however, factors that influence/mediate CYP1A1-activated 4,4'-(hexafluoroisopropylidene)diphenol (BPAF) toxicity, particularly the aryl hydrocarbon receptor (AhR), sulfotransferase (SULT) 1A1 [known to conjugate 2,2-bis(4-hydroxyphenol)-propane (BPA)] and reactive oxygen species (ROS), remain unclear. In this study, a human hepatoma (HepG2) cell line was genetically engineered for the expression of human CYP1A1 and SULT1A1, producing HepG2-hCYP1A1 and HepG2-hSULT1A1, respectively. They were used in the micronucleus test and γ-H2AX analysis (Western blot) (indicating double-strand DNA breaks) with BPAF; the role of AhR in mediating BPAF toxicity was investigated by coexposure of AhR modulators in HepG2 and its derivative C3A (with no genetic modifications but enhanced CYP expression). The results indicated induction of micronuclei by BPAF (≥ 2.5 µM, for 2-cell cycle) in HepG2-hCYP1A1 and C3A, while inactive in HepG2 and HepG2-hSULT1A1; however, BPAF induced micronuclei in HepG2 pretreated with 3,3',4,4',5-pentachlorobiphenyl (PCB126, AhR activator), and BAY-218 (AhR inhibitor) blocked the effect of BPAF in C3A. In HepG2-hCYP1A1 BPAF selectively induced centromere-free micronuclei (immunofluorescent assay) and double-strand DNA breaks. In HepG2 cells receiving conditional medium from BPAF-HepG2-hCYP1A1 incubation micronuclei were formed, while negative in HepG2-hSULT1A1. Finally, the intracellular levels of ROS, superoxide dismutase and reduced glutathione in C3A and HepG2-hCYP1A1 exposed to BPAF were all moderately increased, while unchanged in HepG2 cells. In conclusion, like other BPs BPAF is activated by human CYP1A1 for potent clastogenicity, and this effect is enhanced by AhR while alleviated by SULT1A1.

Involvement of NLRP3/Caspase-1/GSDMD-Dependent pyroptosis in BPA-Induced apoptosis of human neuroblastoma cells.

Bisphenol A (BPA) induces neurotoxicity via enhancing cell apoptosis and inflammation potently (effective at nanomolar concentrations), but its mechanisms remain unidentified. In this study, human neuroblastoma cell lines, IMR-32 and SK-N-SH cells, isolated from a male and a female subject, respectively, were exposed to BPA at various concentrations, with epigallocatechin gallate (EGCG, an antioxidant from green tea), Z-YVAD-FMK (a caspase-1 inhibitor), and ICI182.780 [an estrogen receptor (ER) inhibitor] as modulators. The results showed that BPA increased the mRNA levels of IL-18, ASC, GSDMD and protein levels of NLRP3, caspase-1 and GSDMD in both cell lines in a nonlinear manner. Noticeably, the direction of changes in the mRNA levels of caspase-1 and IL-1ß were opposite, so did each of them in different cell lines: caspase-1 was enhanced in IMR-32 cells but suppressed in SK-N-SH cells, while IL-1ß was suppressed in IMR-32 cells but enhanced in SK-N-SH cells. The level of GSDMD in situ increased along with the leakage of IL-1ß, IL-18, caspase-1 and lactate dehydrogenase (LDH). Moreover, all the above effects of BPA were reversed by Z-YVAD-FMK, ICI182.780, and EGCG. Besides, BPA significantly increased reactive oxygen species production, LDH leakage and apoptosis, with reduced cell viability and mitochondrial membrane potential, in both cell lines, whereas Z-YVAD-FMK and ICI182.780 significantly alleviated the induction of Bak1, Bax, Bcl-2 and caspase-3 proteins by BPA. In summary, BPA may induce pyroptosis in neuroblastoma cells through NLRP3/caspase-1/GSDMD pathway, as mediated by ER; caspase-1-dependent pyroptosis may also contribute to BPA-induced apoptosis, an effect alleviated by EGCG.

Bisphenol F suppresses insulin-stimulated glucose metabolism in adipocytes by inhibiting IRS-1/PI3K/AKT pathway.

Obesity is one of the risk factors of metabolic diseases. Decreased sensitivity to insulin or impairment of the insulin signaling pathway may affect the metabolism of adipose tissue. Bisphenol F (BPF) has been widely used in various products as a substitute for bisphenol A (BPA). BPA has been defined as "obesogen". However, knowledge about the correlation between BPF and obesity is very limited. This study was aimed to explore the effects of BPF on glucose metabolism and insulin sensitivity in mammalian tissues, using a mouse 3T3-L1 adipocyte line as the model. Differentiated 3T3-L1 adipocytes were treated with BPF at various concentrations for 24 h or 48 h, followed by the measurement of cell viability, lipid accumulation, expression levels of adipocytokines, glucose consumption, and impairment of the insulin signaling pathway. The results indicated that BPF had no effect on the size of 3T3-L1 adipocytes, but the expression of leptin, adiponectin and apelin was decreased, while that of chemerin and resistin was increased after 48 h of BPF treatment. Moreover, BPF inhibited the glucose consumption, the expression of GLUT4, and its translocation to the plasma membranes in 3T3-L1 adipocytes. Western blot analysis indicated that the activation of IRS-1/PI3K/AKT signaling pathway was inhibited by BPF, which resulted in reduced GLUT4 translocation. In conclusion, our data suggest that exposure of adipocytes to BPF may alter the expression of calorie metabolism-related adipokines and suppress insulin-stimulated glucose metabolism by impairing the insulin signaling (IRS-1/PI3K/AKT) pathway.

Human CYP enzyme-activated genotoxicity of 2,2',4,4'-tetrabromobiphenyl ether in mammalian cells.

Polybrominated biphenyl ethers (PBDEs) are a group of persistent organic pollutants with endocrine-disrupting, neurotoxic, tumorigenic and DNA-damaging activities. They are hydroxylated by human liver microsomal CYP enzymes, however, their mutagenicity remains unknown. In this study, 2,2',4,4'-tetrabromobiphenyl ether (BDE-47, relatively abundant in human tissues) was investigated for micronuclei induction and DNA damage in mammalian cells. The results indicated that BDE-47 up to 80 µM under a 6 h/18 h (exposure/recovery, covering 2 cell cycles) regime did not induce micronuclei in V79-Mz and V79-derived cell lines expressing human CYP1A1 or 1A2, while it was moderately positive in human CYP2B6-, 2E1-and 3A4-expressing cell lines (V79-hCYP2B6, V79-hCYP2E1-hSULT1A1 and V79-hCYP3A4-hOR, respectively). Following 24 h exposure, BDE-47 induced micronuclei in V79-hCYP2E1-hSULT1A1 and V79-hCYP3A4 cells at increased potencies. In the human hepatoma (HepG2) cells BDE-47 (48 h exposure) was inactive up to 40 µM, however, pretreatment of the cells with ethanol (0.2%, v:v, inducer of CYP2E1) or rifampicin (10 µM, inducer of CYP3A4) led to significant micronuclei formation by BDE-47; pretreatment with bisphenol AF (100 nM) also potentiated BDE-47-induced micronuclei formation (which was blocked by a CYP2E1 inhibitor trans-1,2-dichloroethylene or a CYP3A inhibitor (ketoconazole). Immunofluorescent staining of centromere protein B with the micronuclei formed by BDE-47 in HepG2 cells pretreated with ethanol or rifampicin demonstrated selective formation of centromere-containing micronuclei. The increased phosphorylation of both histones H2AX and H3 in HepG2 by BDE-47 also indicated an aneugenic potential. Therefore, this study suggests that BDE-47 is an aneugen activated by several human CYP enzymes.

Human CYP enzyme-activated clastogenicity of 2-ethylhexyl diphenyl phosphate (a flame retardant) in mammalian cells.

2-Ethylhexyl diphenyl phosphate (EHDPP) is a common flame retardant and environmental pollutant, exposing humans with endocrinal disrupting potentials. Its mutagenicity, especially following metabolism, remains unclear. In this study, molecular docking analysis indicated that EHDPP was a potential substrate for several human CYP enzymes except for CYP1A1. Among V79-derived cell lines genetically engineered for the expression of each CYP, EHDPP (6 h exposure/18 h recovery) did not induce micronuclei in the V79 or V79-derived cells expressing human CYP1A1, however, it was positive in V79-derived cell lines expressing human CYP2E1, 3A4, and 2B6. In a human hepatoma (HepG2) cell line, EHDPP (48 h exposure) moderately induced micronuclei, which was blocked by 1-aminobenzotriazole (ABT, 60 µM, inhibitor of CYPs); pretreating HepG2 cells with bisphenol AF, another organic pollutant as inducer of CYPs (0.1 µM for 16 h), significantly potentiated micronuclei formation by EHDPP, threshold being decreased from 10 to 1.25 µM. This effect was blocked by ABT, drastically reduced by ketoconazole (inhibiting CYP3A expression/activity), and moderately inhibited by trans-1,2-dichloroethylene (selective CYP2E1 inhibitor). Immunofluorescent centromere protein B staining indicated that EHDPP-induced micronuclei in V79-derived cell lines expressing human CYP2E1 and 3A4 were predominantly centromere-negative, and that in HepG2 cells pretreated with bisphenol AF (for inducing multiple CYPs) were purely centromere-negative. In bisphenol AF-pretreated HepG2 cells EHDPP potently induced DNA breaks, as indicated by the comet assay and Western blot analysis of γ-H2AX. In conclusion, our study suggests that EHDPP is potently clastogenic, following activation by several human CYP enzymes, CYP3A4 being a major one.

Influence of Bisphenol Compounds at Nanomolar Concentrations on Chromosome Damage Induced by Metabolically Activated Carcinogens in HepG2 Cells.

Bisphenol (BP) compounds are endocrine-disrupting organic pollutants. BPs may increase the messenger RNA (mRNA) transcripts of nuclear receptors (NRs) regulating the expression of xenobiotic-metabolizing cytochrome P450 (CYP) enzymes. Their impact on the genotoxicity of metabolically activated carcinogens, however, remains unknown. In this study, effects of the bisphenols A, F, S, and AF on the expression of the aryl hydrocarbon receptor (AhR), the pregnane X receptor (PXR), the constitutive androstane receptor, and individual xenobiotic-metabolizing CYP enzymes in a human hepatoma (HepG2) cell line were investigated, along with in silico binding studies of BPs to each receptor. The results indicated that each BP at 1 to 100 nM concentrations increased the mRNA transcripts and protein levels of AhR, PXR, CYP1A1, 1A2, 1B1, 2E1, and 3A4. The predicted affinities of the BPs for binding AhR were comparable to those of potent agonists. Pretreatment of HepG2 cells with each BP potentiated the induction of micronuclei by benzo[a]pyrene, aflatoxin B1, benzene, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; this effect was abolished/reduced by inhibitors of NRs and/or CYPs. Our study suggests that BPs at human exposure levels may aggravate chromosome damage by several impactful carcinogens in human cells by inducing relevant CYP enzymes, mostly via NR modulation.

Bisphenol F promotes the secretion of pro-inflammatory cytokines in macrophages by enhanced glycolysis through PI3K-AKT signaling pathway.

Bisphenol F (BPF) is a member of endocrine disrupting chemicals (EDCs). As a substitute of bisphenol A (BPA), BPF is widely used in various consumer products, leading to an increased risk of people's exposure. However, there are few studies on the immunotoxicity and mechanism of BPF. This study aimed to investigate the effect of BPF on the secretion of pro-inflammatory cytokines by macrophages and explore its mechanism. In our study, RAW264.7 macrophages were treated with different concentrations of BPF (0, 5, 10 and 20 µM) for 24 h. The results showed that the secretion of pro-inflammatory cytokines (IL-6, TNF-α and IL-1ß) and the production of lactate were increased in a dose-dependent manner. BPFalso led to the activation of the PI3K-AKT signaling pathway. After pretreatment with glycolysis inhibitor (2-DG) and exposure to BPF (20 µM), the secretion of pro-inflammatory cytokines induced by BPF was inhibited. PI3K inhibitor (LY294002) and estrogen receptor (ER) antagonist (ICI 182,780) could also inhibit the above effects induced by BPF (20 µM). In conclusion, our results suggested that BPF can enhance glycolysis through ER mediated PI3K-AKT signaling pathway, and the enhanced glycolysis further promoted the secretion of pro-inflammatory cytokines. Our research provides basic data for future studies on bisphenol exposure and immunotoxicity.

Potent aneugenicity of 1-methylpyrene in human cells dependent on metabolic activation by endogenous enzymes.

1-Methylpyrene (1-MP) is a common environmental pollutant and animal carcinogen. After sequential activation by cytochromes P450 and sulfotransferases, it induced gene mutations and micronuclei in mammalian cells. The type of micronuclei formed, entire chromosomes or fragments, was not analysed. In this study, 1-MP and its primary metabolite, 1-hydroxymethylpyrene (1-HMP), were investigated for the induction of centromere-positive and -negative micronuclei in the human hepatoma cell line HepG2 and its derivative C3A, expressing relevant enzymes at higher levels. Under a short-exposure (9 h)/long-recovery regime (2 cell cycles in total), 1-MP and 1-HMP provided negative test results in HepG2 cells. However, they induced micronuclei in C3A cells, the effect being blocked by 1-aminobenzotriazole (inhibitor of cytochromes P450s) and reduced by pentachlorophenol (inhibitor of sulfotransferases). Immunofluorescence staining of centromere protein B in the micronuclei revealed purely clastogenic effects under this regime. Unexpectedly, 1-MP and 1-HMP at concentrations 1/5-1/4 of that required for micronuclei formation led to mitotic arrest and spindle aberrations, as detected by immunofluorescence staining of ß- and γ-tubulin. Following extended exposure (72 h, 2 cell cycles, no recovery), damage to the spindle apparatus and centrosomes was detected at even lower concentrations, with concurrent formation of micronuclei. At low concentrations (1-8 µM 1-MP, 0.25-0.5 µM 1-HMP), the micronuclei induced were unexceptionally centromere-positive. Thus, the chromosome-damaging mechanism of 1-MP was regime and concentration dependent: potently aneugenic under persistent exposure, while clastogenic at higher concentrations following a short-exposure/long-recovery regime. This is a convincing evidence for the existence of metabolic activation-dependent aneugens.

Involvement of a novel regulatory cascade consisting of SET-H3K18ac/H3K27ac-53BP1 in Cr(VI)-induced malignant transformation of 16HBE cells.

Hexavalent chromium (Cr(VI)) is a well-established human carcinogen with DNA damaging effects. Recently we established a Cr(VI)-induced malignant transformation model from a human bronchial epithelial (16HBE) cell line, and in the transformed (16HBE-T) cells reduced levels of 53BP1 (critical for DNA repair) and the acetylated histone H3K18/27 (H3K18/27ac) were observed. In 16HBE-T cells SET (a multifunctional protein) was elevated by Cr(VI) through quantitative proteomics analysis. In the present study, we further explore the involvement of SET in the H3K18/27ac/53BP1 cascade in the 16HBE-T model, primarily by knockdown of SET. Bioinformatic analysis of the differentially expressed proteins indicated enrichment in histone modifications, in which SET was a major regulator. In 16HBE cells SET expression was enhanced by Cr(VI) in a concentration- and exposure duration-dependent manner. In 16HBE-T cells, SET knockdown showed the following effects: reversal of H3K18/27ac and 53BP1 levels, enhanced enrichment H3K18/27ac in 53BP1's promotor region, increase rate of apoptosis and cell cycle G0/G1 arrest (with or without Cr(VI) treatment), and reduced colony-forming efficiency. Finally, In comparison with benzo(a)pyrene-transformed (malignant, 16HBE-B) cells from 16HBE where no changes in H3K18/27ac, 53BP1 or SET were observed, while the H3K18/27ac/53BP1 cascade was downregulated and SET upregulated in 16HBE-T cells, as compared with the parental 16HBE cells; thus the changes in 16HBE-T might be a specific effect of Cr(VI). In conclusion, our results suggest that SET may be involved in the malignant cell transformation, through inhibiting the H3K18/27ac/53BP1 cascade, at least in the 16HBE cell model.